Liposomal clodronate inhibition of osteoclastogenesis and osteoinduction by submicrostructured beta-tricalcium phosphate

Bone graft substitutes such as calcium phosphates are subject to the innate inflammatory reaction, which may bear important consequences for bone regeneration. We speculate that the surface architecture of osteoinductive β-tricalcium phosphate (TCP) stimulates the differentiation of invading monocyte/macrophages into osteoclasts, and that these cells may be essential to ectopic bone formation. To test this, porous TCP cubes with either submicron-scale surface architecture known to induce ectopic bone formation (TCPs, positive control) or micron-scale, non-osteoinductive surface architecture (TCPb, negative control) were subcutaneously implanted on the backs of FVB strain mice for 12 weeks. Additional TCPs samples received local, weekly injections of liposome-encapsulated clodronate (TCPs + LipClod) to deplete invading monocyte/macrophages. TCPs induced osteoclast formation, evident by positive tartrate resistant acid phosphatase (TRAP) cytochemical staining and negative macrophage membrane marker F4/80 immunostaining. No TRAP positive cells were found in TCPb or TCPs + LipClod, only F4/80 positive macrophages and foreign body giant cells. TCPs stimulated subcutaneous bone formation in all implants, while no bone could be found in TCPb or TCPs + LipClod. In agreement, expression of bone and osteoclast gene markers was upregulated in TCPs versus both TCPb and TCPs + LipClod, which were equivalent. In summary, submicron-scale surface structure of TCP induced osteoclastogenesis and ectopic bone formation in a process that is blocked by monocyte/macrophage depletion

Toward osteogenic differentiation of marrow stromal cells and in vitro production of mineralized extracellular matrix onto natural scaffolds

Uncorrected proofTissue engineering has emerged as a new interdisciplinary field for the repair of various tissues, restoring their functions by using scaffolds, cells, and/or bioactive factors. A temporary scaffold acts as an extracellular matrix analog to culture cells and guide the development of new tissue. In this chapter, we discuss the preparation of naturally derived scaffolds of polysaccharide origin, the osteogenic differentiation of mesenchymal stem cells cultured on biomimetic calcium phosphate coatings, and the delivery of biomolecules associated with extracellular matrix mineralization

Influence of ionic strength and carbonate on the Ca-P coating formation from SBF×5 solution

Biomimetic calcium-phosphate (Ca-P) coatings were applied on Ti6Al4V by using simulated body fluids concentrated by a factor 5 (SBF×5). The production of SBF×5 solution was possible by decreasing the pH of the solution to approximately 6 using CO2 gas. The subsequent release of this mildly acidic gas led to a pH rise and thus, increasing supersaturation. After immersion for 5 1/2 h a Ca-P coating on Ti6Al4V plates and a precipitate simultaneously formed at pH=6.8. Sodium chloride (NaCl) and hydrogencarbonate (HCO3−) contents were studied in relation to CO2 release and coating formation by changing their individual concentration in SBF×5 solution. On one hand, NaCl-free or low NaCl-content SBF×5 solution led to the earlier aspecific precipitation in the solution than for SBF×5 solution. In contrast, Ca-P coating was formed later and was thinner than the coating obtained in regular SBF×5 solution. High ionic strength delayed precipitation and favored Ca-P heterogeneous nucleation on Ti6Al4V. On the other hand, HCO3− content increased the pH of the solution due to its buffering capacity and influenced the release rate of dissolved CO2. Thus, HCO3− content strongly affected the supersaturation and Ca-P structure. Furthermore, HCO3− favored the attachment of Ca-P mineral on Ti6Al4V by decreasing Ca-P crystal size resulting in a better physical attachment of Ca-P coating on Ti6Al4V substrate

Biomimetic calcium phosphate coatings on Ti6Al4V: a crystal growth study of octacalcium phosphate and inhibition by Mg2+ and HCO3−

The biomimetic approach for coating metal implants allows the deposition of new calcium phosphate (Ca-P) phases. Films elaborated at physiological conditions might have structures closer to bone mineral than hydroxyl-apatite (HA) plasma-sprayed coatings. In this study, different Ca-P coatings have been deposited through a two-step procedure. After cleaning and etching, Ti6Al4V plates were pretreated by soaking in a simulated body fluid (SBF), i.e., a solution containing inorganic components in concentration more or less similar to body fluids: a thin amorphous carbonated Ca-P layer precipitated on the metal substrate. Second, by soaking these thinly coated metal substrates in another SBF, with different concentrations, the thin amorphous carbonated Ca-P layer led to the fast precipitation of a second and thick Ca-P layer. Different SBF solutions were used in order to investigate the influence of magnesium and carbonate ions. From SBF containing only Ca2+ and HPO42− ions, an octacalcium phosphate layer grew epitaxially on the substrate. When Mg2+ was added into this SBF, the coating was composed of Ca-deficient apatite crystals, while the addition of HCO3− in SBF led to the formation of a B-carbonated apatite layer. Magnesium and carbonate acted as inhibitors of crystal growth. The three phases obtained by our biomimetic process are closer to bone mineral structure than plasma-sprayed HA. Therefore, the obtained results may be particularly relevant for the development of biomimetic Ca-P coatings with optimal bioactivity

Nucleation of biomimetic Ca–P coatings on Ti6Al4V from a SBF×5 solution: influence of magnesium

Biomimetic Calcium–Phosphate (Ca–P) coatings were applied by using 5 times concentrated Simulated Body Fluid (SBF×5) using Carbon Dioxide gas. This process allows the deposition of a uniform Ca–P coating within 24 h. A previous study of our process emphasized the importance of hydrogenocarbonate ions (HCO3−), a crystal growth inhibitor. The aim of the present study was to investigate the role of the other crystal growth inhibitor present in SBF×5, Magnesium (Mg2+), on the Ca–P coating formation. Several SBF×5 solutions were prepared with various Mg2+ and HCO3− contents. No Ca–P deposits were detected on Ti6Al4V substrate soaked for 24 h in a Mg-free SBF×5 solution, whereas by increasing HCO3− content in a Mg-free SBF×5 solution, a Ca–P coating developed on Ti6Al4V substrate. Therefore, it appeared that Mg2+ has a stronger inhibitory effect on apatite crystal growth than HCO3−. Nevertheless, Mg2+ plays also another important role as suggested by depth profile X-ray Photoelectron Spectroscopy (XPS) of the Ca–P coating obtained from SBF×5 solution. Ca2+ and Mg2+ contents increased significantly at the titanium/coating interface. Therefore, Ca2+ and Mg2+ initiated Ca–P coating from SBF×5 solution. The relatively high interfacial concentration in Mg2+ favors heterogeneous nucleation of tiny Ca–P globules onto the substrate. So physical adhesion is enhanced at the early stage of the coating formation

Osteointegration of biomimetic apatite coating applied onto dense and porous metal implants in femurs of goats

Biomimetic calcium phosphate (Ca-P) coatings were applied onto dense titanium alloy (Ti6Al4V) and porous tantalum (Ta) cylinders by immersion into simulated body fluid at 37 °C and then at 50 °C for 24 h. As a result, a homogeneous bone-like carbonated apatitic (BCA) coating, 30 m thick was deposited on the entire surface of the dense and porous implants. Noncoated and BCA-coated implants were press-fit implanted in the femoral diaphysis of 14 adult female goats. Bone contact was measured after implantation for 6, 12, and 24 weeks, and investigated by histology and backscattered electron microscopy (BSEM). After 6 weeks, bone contact of the BCA-coated Ti6Al4V implants was about 50%. After 12 and 24 weeks, bone contact was lower in comparison with the 6-week implantations at, respectively 24 and 39%. Regarding the BCA-coated porous Ta implants, bone contacts were 17, 30, and 18% after 6, 12, and 24 weeks, respectively. However, bone contact was always found significantly higher for BCA-coated dense Ti6Al4V and porous Ta cylinders than the corresponding noncoated implants. The results of this study show that the BCA coating enhances the bone integration as compared to the noncoated implants